The chlorophyll a-binding protein CP47 serves as core antenna to photosystem II (PS II). The predicted topology of CP47 exhibits six membrane-spanning regions and a large hydrophilic loop (loop E) which roughly includes 200 residues (255-455) and is presumably exposed to the lumenal side of the thylakoid membrane. Several lines of experimental evidence suggest that loop E might be involved in binding or stabilizing functional manganese in the catalytic site of water oxidation or in interacting with the extrinsic PS II-O protein (the 33-kDa manganese-stabilizing protein). To scan loop E for functionally important domains, oligonucleotide-directed mutagenesis has been used to introduce deletions of 3-8 residues in conserved and charged regions of loop E. In addition, one single-site mutation of the only histidine present in loop E was created (H343L). Domains deleted in delta 1 (I265-F268), delta 2 (T271-K277), delta 4 (T304-L309), delta 5 (F311-N317), and delta 12 (D440-P447) are required for stable assembly of functional PS II complexes. Deletion of domains delta 3 (K277-E283) and delta 11 (R422-E428) significantly reduces the level of assembled PS II and impairs photoautotrophic growth and oxygen evolution. Deletion of domain delta 8 (A373-D380) enhances the susceptibility to photoinhibition. In contrast, deletion of domains delta 6 (G333-I336), delta 7 (K347-R352), delta 9 (V392-Q394), and delta 10 (D416-F420) and mutation of H343 to leucine do not seem to severly interrupt PS II structure and function, although all mutants exhibit a slightly decreased stability of PS II as compared to the wild type. Thus, selected domains of the large hydrophilic loop of CP47 are important for PS II structure and function. With respect to possible sites of interaction between loop E of CP47 and the extrinsic PS II-O protein, our results indicate that none of the deletions in the region from residue 330 to 420 (delta 6, delta 7, delta 8, delta 9, delta 10) completely interrupts a functional association of the manganese-stabilizing protein to PS II, although the binding characteristics might be changed in some cases.

Oxygen-evolving photosystem I1 core complexes were prepared from spinach by solubilizing photosystem I1 membrane fragments with dodecyl-P-D-maltoside. The core complexes consist of the intrinsic 47-kDa, 43-kDa, D1 and D2 polypeptides, the two subunits of cytochrome b559 and the extrinsic 33-kDa protein. In the presence of 50 mM CaCI2 they exhibit a high oxygen evolution rate of 1.3 0.2 mmol O2 . mg chlorophyll-' . h-' with either 2,6-dichloro-benzoquinone or K,[Fe(CN),] as acceptor. Electron micrographs of these complexes reveal an obtuse triangular structure in when viewed from the top, measuring 15.3 nm on one side and 10.6 nm on the other two sides. An average height of 7.3 nm was determined from the side view position. These data are in good agreement with previously reported dimensions for photosystem 11 core complexes [Irrgang, K.-D., Boekema, E. J., Vater, J. and Renger, G. (1988) Eur. J . Biochem. 178, 209-2171, In contrast to previous reports the extrinsic 33-kDa subunit could be resolved for the first time. It appears as a small protrusion when the complex is viewed from the side and seems to cover the lumenal side of the core complex appearing as a disk with a thickness of 1.5-3.3 nm.In all oxygen-evolving photosynthetic organisms (cyanobacteria, algae and higher plants) the key steps in water oxidation take place in a thylakoid-membrane-bound complex referred to as photosystem 11. The overall reaction sequence of photosystem I1 is energetically driven by photooxidation of a special chlorophyll a (P680), with pheophytin as the primary acceptor, and subsequent electron transfer to a specifically bound plastoquinone molecule (QA). The latter step is required for stabilization of the primary charge separation sufficient to permit water cleavage [l]. The holes produced at P680' lead to oxidation of water to dioxygen via a four-step univalent redox-reaction sequence at a manganesecontaining catalytic site; whereas the electrons of QA reduce mobile plastoquinone to the quinol form via a two-step univalent mechanism at a specific binding site denoted as the Qs site (for recent reviews see [2 -41). Based on different lines of evidence, all functional redox groups of photosystem I1 are currently assumed to be incorporated into a heterodimer of two polypeptides designated as D1 and D2, each consisting of about 350 amino acid residues. This assignment would imply that water oxidation would also occur within this complex. D1 and D2 exhibit remarkable sequence analogies with the subunits L and M of purple-bacteria reaction centers (for review see [5, 6]), but in contrast to the situation for purplebacteria reaction centers, the detailed structure of the D1/D2 heterodimer remains unresolved.One key problem is the failure to develop a suitable procedure which permits the preparation of functionally active D1 /D2 complex. All photosystem I1 reaction center complexes isolated so far contain, in addition to D1/D2, the two subunits of cytochrome b559 and probably at least one further subunit of small relative molecular ...

Several autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions or a single-site mutation within the large, lumen-exposed loop (loop E) of the chlorophyll a-binding photosystem II core protein, CP47, are analyzed for their functional properties by measuring the flash-induced pattern of thermoluminescence, oxygen yield, and fluorescence quantum yield. A physiological and biochemical characterization of these mutant strains has been given in two previous reports [Eaton-Rye, J.J., & Vermaas, W.F.J. (1991) Plant Mol. Biol. 17, 1165-1177; Haag, E., Eaton-Rye, J.J., Renger, G., & Vermaas, S. F.J. (1993) Biochemistry 32, 4444-4454]. The results of the present study show that deletion of charged and conserved amino acids in a region roughly located between residues 370 and 390 decreases the binding affinity of the extrinsic PS II-O protein to photosystem II. Marked differences with PSII-O deletion mutants are observed with respect to Ca2+ requirement and the flash-induced pattern of oxygen evolution. Under conditions where a sufficient light activation is provided, the psbB mutants assayed in this study reveal normal S-state parameters and lifetimes. The results bear two basic implications: (i) the manganese involved in water oxidation can still be bound in a functionally normal or only slightly distorted manner, and (ii) the binding of the extrinsic PS II-O protein to photosystem II is impaired in mutants carrying a deletion in the domain between residues 370 and 390, but the presence of the PS II-O protein is still of functional relevance for the PS II complex, e.g., for maintenance of a high-affinity binding site for Ca2+ and/or involvement during the process of photoactivation.

Studies on thermodynamics and kinetics of electron transfer from QA- to QB(QB-) were performed by monitoring laser flash induced changes of the relative fluorescence emission as a function of temperature (220 K < T < 310 K) in isolated thylakoids and PS II membrane fragments.In addition, effects of bivalent metal ions on PS II were investigated by measuring conventional fluorescence induction curves, oxygen evolution, manganese content and atrazine binding mostly in PS II membrane fragments. It was found: a) the normalized level of the fluorescence remaining 10 s after the actinic flash (Ft/F0) steeply increases at temperatures below -10 to - 20 °C, b) the fast phase of the transient fluorescence change becomes markedly retarded with decreasing temperatures, c) among different cations (Cu2+, Zn2+, Cd2+, Ni2+, Co2+) only Cu2+ exhibits marked effects in the concentration range below 100 μᴍ and d) Cu2+ decreases the normalized variable fluorescence, inhibits oxygen evolution and diminishes the affinity to atrazine binding without affecting the number of binding sites. The content of about four manganeses per functionally competent oxygen evolving complex is not changed by [Cu2+] < 70 μᴍ.Based on these findings it is concluded: i) a temperature dependent equilibrium between an inactive (I) and active (A) state of QA- reoxidation by QB(QB- ) is characterized by standard enthalpies ΔH° of 95 kJ mol-1 and 60 kJ mol-1 and standard entropies ΔS° of 370 kJ K-1 mol-1 and 240 kJ K-1 mol-1 in isolated thylakoids and PS II membrane fragments, respectively, ii) the activation energies of QA- reoxidation by plastoquinone bound to the QB site are about 30 kJ mol-1 (thylakoids) and 40 kJ mol-1 (PS II membrane fragments) in 220 K < T < 300 K, and iii) Cu2+ causes at least a two-fold effect on PS II by modifying the atrazine binding affinity at lower concentrations ( ~ 5 μᴍ) and interference with the redox active tyrosine Yz at slightly higher concentration ( ~ 10 μᴍ) leading to blockage of oxygen evolution.

Oscillation patterns of the oxygen yield per flash induced by a train of single-turnover flashes were measured as a function of dark incubation and different pre-illumination conditions in several autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions within the large, lumen-exposed hydrophilic region (loop E) of the chlorophyll a-binding photosystem II protein CP47. A physiological and biochemical characterization of these mutant strains has been presented previously [Eaton-Rye, J. J., & Vermaas, W. F. J. (1991) Plant Mol. Biol. 17, 1165-1177; Haag, E., Eaton-Rye, J. J., Renger, G., & Vermaas, W. F. J. (1993) Biochemistry 32, 4444-4454], and some functional properties were described recently [Gleiter, H. M., Haag, E., Shen, J.-R., Eaton-Rye, J. J., Inoue, Y., Vermaas, W. F. J., & Renger, G. (1994) Biochemistry 33, 12063-12071]. The present study shows that in several mutants the water-oxidizing complex (WOC) became inactivated during prolonged dark incubation, whereas the WOC of the wild-type strain remained active. The rate and extent of the inactivation in the mutants depend on the domain of loop E, where 3-8 amino acid residues were deleted. The most pronounced effects are observed in mutants delta(A373-D380) and delta(R384-V392). A competent WOC can be restored from the fully inactivated state by illumination with short saturating flashes. The number of flashes required for this process strongly depends on the site at which a deletion has been introduced into loop E. Again, the most prominent effects were found in mutants delta(A373-D380) and delta(R384-V392). Interestingly, the number of flashes required for activation was reduced by more than an order of magnitude in both mutants by the addition of 10 mM CaCl2 to the cell suspension. On the basis of a model for photoactivation proposed by Tamura and Cheniae (1987) [Biochim. Biophys. Acta 890, 179-194], a scheme is presented for the processes of dark inactivation and photoactivation in these mutants. The results presented here corroborate an important role of the large hydrophilic domain (loop E) of CP47 in a functional and stable WOC.

The functional properties of a purified homogeneous spinach PS II-core complex with high oxygen evolution capacity (Haag et al. 1990a) were investigated in detail by measuring thermoluminescence and oscillation patterns of flash induced oxygen evolution and fluorescence quantum yield changes. The following results were obtained: a) Depending on the illumination conditions the PS II-core complexes exhibit several thermoluminescence bands corresponding to the A band, Q band and Zv band in PS II membrane fragments. The lifetime of the Q band (Tmax=10°C) was determined to be 8s at T=10°C. No B band corresponding to S2QB (-) or S3QB (-) recombination could be detected. b) The flash induced transient fluorescence quantum yield changes exhibit a multiphasi relaxation kinetics shich reflect the reoxidation of Q A (-) . In control samples without exogenous acceptors this process is markedly slower than in PS II membrane fragments. The reaction becomes significantly retarded by addition of 10 μM DCMU. After dark incubation in the presence of K3[Fe(CN)6 c) Excitation of dark-adapted samples with a train of short saturating flashes gives rise to a typical pattern dominated by a high O2 yield due to the third flash and a highly damped period four oscillation. The decay of redox states S2 and S3 are dominated by short life times of 4.3 s and 1.5 s, respectively, at 20°C. The results of the present study reveal that in purified homogeneous PS II-core complexes with high oxygen evolution isolated from higher plants by β-dodecylmaltoside solubilization the thermodynamic properties and the kinetic parameters of the redox groups leading to electron transfer from water to QA are well preserved. The most obvious phenomenon is a severe modification of the QB binding site. The implications of this finding are discussed.

Effects of photoinhibition at 0 °C on the PS II acceptor side have been analyzed by comparative studies in isolated thylakoids, PS II membrane fragments and PS II core complexes from spinach under conditions where degradation of polypeptide(s) D1(D2) is highly retarded. The following results were obtained by measurements of the transient fluorescence quantum and oxygen yield, respectively, induced by a train of short flashes in dark-adapted samples: (a) in the control the decay of the fluorescence quantum yield is very rapid after the first flash, if the dark incubation was performed in the presence of 300 μM K3[Fe(CN)6]; whereas, a characteristic binary oscillation was observed in the presence of 100 μM phenyl-p-benzoquinone with a very fast relaxation after the even flashes (2nd, 4th. . . ) of the sequence; (b) illumination of the samples in the presence of K3[Fe(CN)6] for only 5 min with white light (180 W m(-2)) largely eliminates the very fast fluorescence decay after the first flash due to QA (-) reoxidation by preoxidized endogenous non-heme Fe(3+), while a smaller effect arises on the relaxation kinetics of the fluorescence transients induced by the subsequent flashes; (c) the extent of the normalized variable fluorescence due to the second (and subsequent) flash(es) declines in all sample types with a biphasic time dependence at longer illumination. The decay times of the fast (6-9 min) and the slow degradation component (60-75 min) are practically independent of the absence or presence of K3[Fe(CN)6] and of anaerobic and aerobic conditions during the photo-inhibitory treatment, while the relative extent of the fast decay component is higher under anaerobic conditions. (d) The relaxation kinetics of the variable fluorescence induced by the second (and subsequent) flash(es) become retarded due to photoinhibition, and (e) the oscillation pattern of the oxygen yield caused by a flash train is not drastically changed due to photoinhibition.Based on these findings, it is concluded that photoinhibition modifies the reaction pattern of the PS II acceptor side prior to protein degradation. The endogenous high spin Fe(2+) located between QA and QB is shown to become highly susceptible to modification by photoinhibition in the presence of K3[Fe(CN)6] (and other exogenous acceptors), while the rate constant of QA (-) reoxidation by QB(QB (-)) and other acceptors (except the special reaction via Fe(3+)) is markedly less affected by a short photoinhibition. The equilibrium constant between QA (-) and QB(QB (-)) is not drastically changed as reflected by the damping parameters of the oscillation pattern of oxygen evolution.

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